Protein lysine methylation plays an important role in histone-mediated control of gene function. Histone methylation is mediated by distinct histone methyltransferases that play an important role in cell differentiation and proliferation. The potent role of histone methylation in regulation of their function raises questions about the possible use of protein lysine methylation in regulation of non-histone protein function. Recently, we found that methyltransferase Ezh2, which has been implicated in chromatin-mediated control of cell differentiation, possesses previously unknown signaling function in the T cell cytosol. Using conditional Ezh2 inactivation we showed the essential role of Ezh2 in regulation of T cell receptor-mediated actin polymerization and cell activation. We hypothesize that Ezh2 controls T cell signaling through lysine methylation of signaling proteins involved in actin polymerization. In the current proposal, we describe the experiments that will employ genetic and biochemical approaches to reveal the mechanism of Ezh2 cytosolic signaling and its significance in T cell development and responses. We will test whether and how Ezh2's association with Vav1, a key signal transducer in T cells, contributes to T cell signaling and function. Using methods of conditional mutagenesis in vivo, we will address the ability of cytosolic Ezh2 to mediate signaling processes that govern T cell development and activation. We also will provide evidence for the existence of the cytosolic Ezh2 substrate and outline the strategy for its characterization and functional analysis. Overall, understanding of Ezh2 signaling may help to identify a fundamentally novel signaling pathway that plays an important role in regulation of signaling in T cells, as well as other cell types. The proposed experiments have the potential to identify previously unknown signaling mechanisms that an essential role in activation of T cells as well as other cell types. The relevance of the proposed project to public health is in that the identification of a novel signaling pathway could be used for the purposes of immunomodulation and cancer therapy.